Extracellular Domain Of Transmembrane Proteins Research Articles

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Human Coxsackie- and adenovirus receptor is a putative target of neutrophil elastase-mediated shedding

BackgroundDuring viral-induced myocarditis, immune cells migrate towards the site of infection and secrete proteases, which in turn can act as sheddases by cleaving extracellular domains of transmembrane proteins. We were interested in the shedding of the Coxsackie- and adenovirus receptor (CAR) that acts as an entry receptor for both eponymous viruses, which cause myocarditis. CAR shedding by secreted immune proteases could result in a favourable outcome of myocarditis as CAR’s extracellular domain would be removed from the cardiomyocytes’ surface leading to decreased susceptibility to ongoing viral infections.Methods and resultsIn this work, matrix metalloproteinases and serine proteinases were screened for their proteolytic activity towards human CAR. Whereas matrix metalloproteinases, proteinase 3, and cathepsin G did not cleave human recombinant CAR or only within long incubation times, neutrophil elastase showed a distinct cleavage pattern of CAR’s extracellular domain that was time- and dose-dependent. Neutrophil elastase cleaves CAR at its membrane-proximal immunoglobulin domain as we determined by nanoLC-MS/MS. Furthermore, neutrophil elastase treatment of cells reduced CAR surface levels as seen by flow cytometry and immunofluorescence microscopy.ConclusionsWith this study, we show that CAR might be a target for shedding by neutrophil elastase.

Ligand Selection by Combination of Recombinant and Cell Panning Selection Techniques.

High-throughput protein selection methods are a cornerstone for protein engineering and pharmaceutical development. Traditional high-throughput selection strategies rely largely on recombinant antigen to generate target specificity. Though effective, this selection strategy can be limited by soluble target quality, particularly in the case of recombinant extracellular domains of transmembrane proteins. Recent advances in cell-based selection techniques provide new opportunities for improving the outcomes of ligand selection campaigns but can introduce technical challenges in maintaining antigen specificity due to the heterogeneity of biomacromolecule expression on the mammalian cell surface. Here, we describe a combination technique using recombinant antigen to "train" library target specificity followed by cell panning selections to ensure that isolated ligands bind cell-expressed target, as well as a facile microscopy technique for assessing target specificity on a clonal basis without the need to produce soluble ligand.

Assessment of Scavenger Receptor Cysteine-Rich Domain Binding to Bacteria.

The scavenger receptor cysteine-rich SRCR domain is an ancient protein domain found in SR-A and SR-I scavenger receptors, which is characterized by a conserved arrangement of cysteines (Martinez et al., Pharmacol Rev 63(4):967-1000, 2011; Sarrias et al., Crit Rev Immunol 24(1):1-37, 2004; Telfer and Baldwin, Cell Immunol 296(1):76-86, 2015; PrabhuDas et al., J Immunol, 2017. 198(10):3775-3789). SRCR domains are divided into group A and group B SRCR domains by virtue of how many cysteines they contain and the resulting disulfide bonding pattern. Group B SRCR domains, found in WC1, CD163, CD5, CD6, Spα and DMBT1, are approximately 100-110 amino acids long and contain 6-8 cysteines predicted to form 3-4 disulfide bonds. The crystal structure of a CD5 group B SRCR domain predicts a fold of two beta-sheets and an alpha helix (Rodamilans et al., J Biol Chem 282(17):12669-12677, 2007; Wang et al., Mol Immunol 48:801-809, 2011). SRCR domains bind to many different types of chemical compounds found on cells, viruses, and microbes and are usually found in multiples in the extracellular domains of transmembrane proteins or in secreted proteins. Small amino acid differences between these SRCR domains lead to significant differences in binding affinity. In addition, SRCR domain genes contain allelic polymorphisms and can be extensively duplicated. Thus, single and duplicated SRCR domain protein gene loci encode a large tunable binding potential. Binding to pathogen-associated molecular patterns (PAMPs) combined with signaling potential predicts an important role for these molecules in the immune response. WC1 SRCR domains bind to the spirochetes Leptospira and Borrelia (Hsu et al., J Immunol 194(5):2280-2288, 2015). CD6 (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007), Spα (Sarrias et al., J Biol Chem 280(42):35391-35398, 2005), CD163A (Fabriek et al., Blood 113(4):887-892, 2009) and DMBT1 (Madsen et al., Eur J Immunol 33(8):2327-2336, 2003) bind to Gram-positive and Gram-negative bacteria; CD5 binds to yeast (Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009). Identified ligands include lipoteichoic acid, lipopolysaccharide, poly-phosphorylated, and -sulfated compounds such as dextran sulfate sodium, leucine-rich repeat proteins, and fungal mannose (Sarrias et al., Proc Natl Acad Sci U S A 104(28):11724-11729, 2007; Sarrias et al., J Biol Chem 280(42):35391-35398, 2005; Fabriek et al., Blood 113(4):887-892, 2009; Vera et al., Proc Natl Acad Sci U S A 106(5):1506-1511, 2009; End et al., Eur J Immunol 39(3):833-842, 2009; Loimaranta et al., J Biol Chem 284(28):18614-18623, 2009). A conserved linear binding motif (VEVLXXXXW) in an external loop in the SRCR domain has been identified in CD163A and DMBT1 and can be used as a peptide that aggregates bacteria (Fabriek et al., Blood 113(4):887-892, 2009; Bikker et al., J Biol Chem 279(46):47699-47703, 2004; Leito et al., Biol Chem 389(9):1193-1200, 2008). In contrast, WC1 binding to bacteria is mediated by a noncontinuous motif in the native protein, and mutation of the VEVLXXXXW motif has no effect upon bacterial binding (Hsu et al., J Immunol 194(5):2280-2288, 2015). Thus, bacterial binding studies with WC1 SRCR domains must be done with native, correctly disulfide bonded, protein, ideally posttranslationally modified in mammalian cells.WC1 is found in the genomes of most mammals, reptiles, and birds and is expressed exclusively on γδ T cells in ruminants. The 13 bovine WC1 genes encode up to 11 extracellular SRCR domains, organized in the SRCR domain pattern of a1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d'11], where the alphabet designations indicate homology between genes and across species (Chen et al., BMC Genet 13:86, 2012; Herzig et al., BMC Evol Biol 10:181, 2010; Herzig and Baldwin, BMC Genomics 10:191, 2009). Some of the signaling co-receptor WC1 molecules are required for the γδ T cell response to Leptospira (Wang et al., Mol Immunol 48:801-809, 2011; Rogers et al., J Immunol 174(6):3386-3393, 2005; Wang et al., Eur J Immunol 39(1):254-266, 2009). The WC1 expressed on responsive γδ T cells is correlated with its direct binding to Leptospira via some of its SRCR domains (Hsu et al., J Immunol 194(5):2280-2288, 2015). Because WC1+ γδ T cells share a restriction in their γδ TCRs and WC1 has TCR co-receptor activity, we hypothesize that WC1 co-ligation with the TCR plays the determining role in the activation of WC1+ γδ T cells by pathogens. Classification of the binding of WC1 SRCR domains, their ligands, and their role in the interaction of 𝛾δ T cells with pathogens relevant to the host will allow these cells to be recruited in next-generation vaccines to pathogens that have significant negative economic and health impact.

The Secreted Tyrosine Kinase Vlk Is Essential for Normal Platelet Activation and Thrombus Formation

Tyrosine phosphorylation of proteins secreted into the extracellular space has been observed in cell cultures and in vivo, yet little is known about the role that phosphorylation of extracellular proteins serves in modulating cell function. An important reason for the gap in our knowledge of the functional significance of extracellular protein phosphorylation has been the delay in identifying extracellular kinases. Within the last decade, however, bioinformatic strategies to identify kinases with signal peptides, coupled with biochemical approaches to characterize kinases in the secretory pathway, have described several kinases that phosphorylate secretory pathway and extracellular substrates. Of the known kinases containing signal sequences, Fam20B and VLK have been identified in platelets. VLK has been identified as a broadly expressed secretory pathway tyrosine kinase secreted from platelets in an activation dependent manner. Its role in platelet function, however, has not been previously studied. To understand the contribution of tyrosine phosphorylation of secreted factors and extracellular domains of transmembrane proteins in platelet function and thrombus formation, we generated mice whose platelets lacked VLK. Mice with megakaryocyte/platelet-specific VLK deficiency (Vlk-cKO) exhibited normal platelet abundance, volume and morphology, and tail clip bleeding times, but showed dramatic changes in platelet function in vitro and in vivo. In vivo, platelet accumulation was reduced by 90% in Vlk-cKO mice compared to control (Vlkf/f) littermates (P = 0.02) following laser-induced injury of cremaster arterioles (Figure). Likewise, fibrin generation was reduced in mice lacking platelet VLK by 62% (P = 0.009). In vitro, evaluation of resting and thrombin-stimulated VLK-deficient platelets demonstrated a significant decrease of several tyrosine phosphobands compared to control. Platelet function testing of VLK-deficient platelets (Figure) showed decreased platelet aggregation in response to stimulation with 100 µM AYPGKF, a PAR4 agonist, (Vlkf/f: 70+5.1%; Vlk-cKO: 23+8.0%) or 4 µg/mL collagen (Vlkf/f: 53+2.5%; Vlk-cKO: 27.5+2.9%). Dense and α-granule release in response to AYPGKF were also significantly decreased in platelets in which VLK had been silenced. In contrast, Vlk-cKO platelets aggregated normally in response to either 10 µM, 40 µM, or 100 µM ADP, and the aggregation defect in response to low doses of AYPGKF was reversed by subthreshold concentrations (2.5 µM) of ADP. Furthermore, stimulation with high-dose 150 µM AYPGFK or 5 U/ml thrombin resulted in comparable platelet function and ATP secretion in control and Vlk-cKO platelets respectively, ruling out a storage pool defect. Taken together, these results suggest that a dense granule secretion defect contributes to the decrease in platelet aggregation observed in platelets in which VLK is absent. In human platelets, tyrosines phosphorylated in secreted and extracellular domains of transmembrane proteins implicated in the regulation of platelet function were identified by mass spectroscopy analysis. Extracellular proteins or proteins with phosphosites that mapped to extracellular domains included ectonucleoside triphosphate diphosphodydrolase 6 [ENTPD6], platelet basic protein, integrin αIIß, and multimerin-1. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus dependent platelet aggregation and thrombus formation, and provide the first evidence that secreted kinases contribute to platelet function. Disclosures De Ceunynck: Sanofi: Current Employment. Dilks:PlateletBiogenesis: Current Employment. Peters:PlateletBiogenesis: Current Employment. Noetzli:Anylam: Current Employment. Rosen:Keros Therapeutics: Membership on an entity's Board of Directors or advisory committees. Italiano:PlateletBioGenesis: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Flaumenhaft:STRM.Bio: Membership on an entity's Board of Directors or advisory committees; PlateletDiagnostics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; QuercisPharma: Membership on an entity's Board of Directors or advisory committees.

ADAM10: a new player in breast cancer progression?

Background:The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes.Methods:ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration.Results:Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (P<0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, P<0.001; for MDA-MB-231, P=0.005; for MDA-MB-453, P=0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (P<0.001) and in oestrogen receptor (ER)-negative compared with ER-positive tumours (P=0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer.Conclusions:Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype.

Proteomic Identification of Cysteine Cathepsin Substrates Shed from the Surface of Cancer Cells

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192.

The multiple roles of epidermal growth factor repeat O-glycans in animal development.

The epidermal growth factor (EGF)-like repeat is a common, evolutionarily conserved motif found in secreted proteins and the extracellular domain of transmembrane proteins. EGF repeats harbor six cysteine residues which form three disulfide bonds and help generate the three-dimensional structure of the EGF repeat. A subset of EGF repeats harbor consensus sequences for the addition of one or more specific O-glycans, which are initiated by O-glucose, O-fucose or O-N-acetylglucosamine. These glycans are relatively rare compared to mucin-type O-glycans. However, genetic experiments in model organisms and cell-based assays indicate that at least some of the glycosyltransferases involved in the addition of O-glycans to EGF repeats play important roles in animal development. These studies, combined with state-of-the-art biochemical and structural biology experiments have started to provide an in-depth picture of how these glycans regulate the function of the proteins to which they are linked. In this review, we will discuss the biological roles assigned to EGF repeat O-glycans and the corresponding glycosyltransferases. Since Notch receptors are the best studied proteins with biologically-relevant O-glycans on EGF repeats, a significant part of this review is devoted to the role of these glycans in the regulation of the Notch signaling pathway. We also discuss recently identified proteins other than Notch which depend on EGF repeat glycans to function properly. Several glycosyltransferases involved in the addition or elongation of O-glycans on EGF repeats are mutated in human diseases. Therefore, mechanistic understanding of the functional roles of these carbohydrate modifications is of interest from both basic science and translational perspectives.

Proteomic database mining opens up avenues utilizing extracellular protein phosphorylation for novel therapeutic applications.

SummaryRecent advances in extracellular signaling suggest that extracellular protein phosphorylation is a regulatory mechanism outside the cell. The list of reported active extracellular protein kinases and phosphatases is growing, and phosphorylation of an increasing number of extracellular matrix molecules and extracellular domains of trans-membrane proteins is being documented. Here, we use public proteomic databases, collagens – the major components of the extracellular matrix, extracellular signaling molecules and proteolytic enzymes as examples to assess what the roles of extracellular protein phosphorylation may be in health and disease. We propose that novel tools be developed to help assess the role of extracellular protein phosphorylation and translate the findings for biomedical applications. Furthermore, we suggest that the phosphorylation state of extracellular matrix components as well as the presence of extracellular kinases be taken into account when designing translational medical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0482-4) contains supplementary material, which is available to authorized users.

Abstract 4212: ADAM10/Kuzbanian is upregulated during neuroendocrine prostate carcinogenesis

Abstract Prostate cancer (CaP) is the second leading cause of cancer death in men. Advanced prostate tumors often contain neuroendocrine differentiation, which correlates with androgen-independent progression and poor prognosis. Currently, androgen deprivation is the first line of therapy for metastatic CaP. However, CaP often progresses to an androgen-independent bone-metastatic stage, at which point chemotherapy and radiotherapy become the primary therapeutic options. Despite recent advances in therapeutic strategies, many cancers still develop resistance to radiation and therapies, making the identification of new therapies essential to improve the lives and survival rates of patients. We have studied neuroendocrine cancer progression in the cryptdin-2 SV40-TAg (CR2-TAg) mouse model of CaP. Since these cancers do not express androgen receptor, they are refractory to androgen therapy. Therefore, we sought new potential targets. We previously found that matrix metalloproteinases (MMPs), which are capable of degrading many extracellular matrix proteins and cell surface receptors, are upregulated during CR2-Tag tumor progression. In the present study, we examined expression of a family of a disintegrin and metalloproteinases (ADAMs) that is actively involved in these signaling pathways due to their role in releasing the extracellular domain of transmembrane proteins. ADAMs contain features of both adhesive proteins and proteinases. ADAM10 activates the epidermal growth factor receptor (EGFR) and NOTCH signaling systems controlling cell growth, invasion, and metastasis. We found that ADAM10 expression increased during tumor progression and was highly penetrant. We are quantifying the expression levels and localization patterns of ADAM10 in these samples over time. Since MMPs, like ADAMs, are capable of degrading many extracellular matrix proteins and cell surface receptors, we determined if MMPs (MMP 2, 7, and 9) contribute to ADAM10 expression or localization in aggressive hormone refractory CaP. We have stained for ADAM10 in tissue sections from the CR2-TAg mice that are each missing expression of MMPs to determine if that particular MMP contributes to ADAM10 expression or localization. Our research using ADAM10 localization as a potential biomarker and therapeutic target has potential clinical value to identify aggressive CaP earlier in patients. Our study of MMPs and their role in ADAM10 expression will offer insight into the mechanisms of advanced prostate tumor progression. Note: This abstract was not presented at the meeting. Citation Format: Brian D. Shannon, Laurie E. Littlepage. ADAM10/Kuzbanian is upregulated during neuroendocrine prostate carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4212. doi:10.1158/1538-7445.AM2014-4212

Metalloproteinases in Corneal Diseases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with the potential to degrade all types of extracellular matrix. The ADAM (a disintegrin and metalloproteinase) family of peptidases was recently identified as cleaving the extracellular domain of transmembrane proteins. This was termed ectodomain shedding. We investigated the MMP expression in patients with corneal diseases and the potential role of ADAMs in corneal pathophysiology. We detected upregulation of the active form of MMP-2 and MMP-9 in the tear fluid from patients with corneal melting or recurrent corneal erosion. Using human corneal epithelial cells, we observed ADAM17-dependent ectodomain shedding of soluble tumor necrosis factor receptor 1 and soluble interleukin-6 (IL-6) receptor (sIL-6R). The production of sIL-6R was also induced by messenger RNA splicing in the human corneal epithelial cells. IL-6/sIL-6R-induced signal transducer and activator of transcription 3 phosphorylation was observed in cultured human corneal fibroblasts, suggesting that IL-6 trans-signaling induced inflammatory cellular signaling in the human corneal fibroblasts. We demonstrated that MMPs are significantly upregulated in collagen-destructive disorders of the cornea. Additionally, we observed that ectodomain shedding by ADAMs in corneal epithelial cells mediated the production of soluble cytokine receptors. Trans-signaling of IL-6 can induce an inflammatory response in corneal stroma, indicating the significance of IL-6 trans-signaling in ocular surface inflammation. Thus, MMPs and ADAMs play an important role in the pathophysiology of corneal diseases.

ADAM10 Regulates Transcription Factor Expression Required for Plasma Cell Function

A disintegrin and metalloprotease 10 (ADAM10) is a key regulator of cellular processes by shedding extracellular domains of transmembrane proteins. We have previously demonstrated that deletion of B cell expressed ADAM10 results in changes in lymphoid tissue architecture and impaired germinal center (GC) formation. In this study, mice were generated in which ADAM10 is deleted in B cells following class switch recombination (ADAM10Δ/ΔIgG1-cre+/− mice). Despite normal GC formation, antibody responses were impaired in ADAM10Δ/ΔIgG1-cre+/− mice, implicating ADAM10 in post-GC and extrafollicular B cell terminal differentiation. Surprisingly, plasma cell (PC) numbers were normal in ADAM10Δ/ΔIgG1-cre+/− mice when compared to controls. However, PCs isolated from ADAM10Δ/ΔIgG1-cre+/− mice exhibited decreased expression of transcription factors important for PC function: Prdm1, Xbp1 and Irf4. Bcl6 is a GC transcriptional repressor that inhibits the PC transcriptional program and thus must be downregulated for PC differentiation to occur. Bcl6 expression was increased in PCs isolated from ADAM10Δ/ΔIgG1-cre+/− mice at both the mRNA and protein level. These results demonstrate that ADAM10 is required for proper transcription factor expression in PCs and thus, for normal PC function.

Abstract 1845: ADAM10: A new player in breast cancer progression

Abstract Background: The ADAMs are a family of proteases, best known for their role in releasing the extracellular domain of transmembrane proteins. One of the ADAMs, i.e., ADAM10, is involved in the activation of 2 important signaling systems controlling cell growth, invasion and metastasis, i.e. the EGFR and NOTCH signaling systems. Because of its ability to activate both EGFR and NOTCH, ADAM10 is likely to play a role in cancer formation or progression. The aim of this study was therefore to investigate the role of ADAM10 in breast cancer and test its therapeutic potential, especially in patients with the triple-negative form of this disease. Materials and Methods: ADAM10 mRNA expression was examined in a pooled collection of publically available databases (n ∼ 4000). ADAM10 protein was examined in a cohort of 120 breast tumor extracts by ELISA. In addition, ADAM10 expression was decreased by RNAi and the effects of this on cell proliferation, invasion and migration were determined. Using the novel ADAM10 inhibitor GI254023X (GSK), we examined the effect of ADAM10 inhibition on a panel of 13 breast cancer cell lines. Results: Using the pooled collection of databases, ADAM10 mRNA was found to be significantly elevated in HER2+ breast cancer compared to other subtypes (p &amp;lt; 0.0001). High ADAM10 mRNA expression was also observed in basal like breast cancer and was associated with poorer overall survival in this subgroup (p = 0.0196). In a cohort of 120 breast tumor extracts, ADAM10 protein was found to be significantly higher in ER-negative compared to ER-positive tumors (p = 0.005), in high grade versus low grade tumors (p &amp;lt; 0.0001), and in younger than older women (p = 0.018). Downregulation of ADAM10 in MDA-MB-231 breast cancer cells resulted in a significant reduction in invasion (p = 0.0006) and cellular migration (p = 0.0002). Treatment of 13 breast cancer cell lines with GI254023X resulted in variable growth inhibition (from 0 to 52%). Furthermore, GI254023X significantly decreased invasion in the MDA-MB-231 cell line (p = 0.001). Conclusions: Our findings of a correlation between ADAM10 and features of aggressive disease and the fact that its downregulation/inhibition decreased growth, invasion and migration suggests that this ADAM10 protease is involved in the progression of breast cancer. Inhibition of ADAM10 with GI254023X may be a new treatment for breast cancer. Acknowledgements: The authors thank SFI and the Molecular Therapeutics for Cancer Ireland (SRC award, 08/SRC/B1410 to MTCI) for funding this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1845. doi:1538-7445.AM2012-1845

Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras

The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation-resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady-state levels and is compatible with kinetic analysis of endocytosis in live cells.

Prediction of protein–protein interactions between Helicobacter pylori and a human host

A lack of information on protein-protein interactions at the host-pathogen interface is impeding the understanding of the pathogenesis process. A recently developed, homology search-based method to predict protein-protein interactions is applied to the gastric pathogen, Helicobacter pylori to predict the interactions between proteins of H. pylori and human proteins in vitro. Many of the predicted interactions could potentially occur between the pathogen and its human host during pathogenesis as we focused mainly on the H. pylori proteins that have a transmembrane region or are encoded in the pathogenic island and those which are known to be secreted into the human host. By applying the homology search approach to protein-protein interaction databases DIP and iPfam, we could predict in vitro interactions for a total of 623 H. pylori proteins with 6559 human proteins. The predicted interactions include 549 hypothetical proteins of as yet unknown function encoded in the H. pylori genome and 13 experimentally verified secreted proteins. We have recognized 833 interactions involving the extracellular domains of transmembrane proteins of H. pylori. Structural analysis of some of the examples reveals that the interaction predicted by us is consistent with the structural compatibility of binding partners. Examples of interactions with discernible biological relevance are discussed.

The Single Subunit Transmembrane E3 Ligase Gene Related to Anergy in Lymphocytes (GRAIL) Captures and Then Ubiquitinates Transmembrane Proteins across the Cell Membrane

The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface down-regulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.

Discovery of a Cytokine and Its Receptor by Functional Screening of the Extracellular Proteome

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.

Impaired Trafficking and Activation of Tumor Necrosis Factor-α-converting Enzyme in Cell Mutants Defective in Protein Ectodomain Shedding

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.

Maleimide-functionalized lipids that anchor polypeptides to lipid bilayers and membranes.

Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.